1. Field of the Invention
Compounds are provided herein that bind to Bcl-2-family member peptides and alter their apoptosis regulatory function. More specifically, the use of peptides expressed by the TR3 gene or chemical compounds that mimic the effects of TR3 to induce Bcl-2 or Bcl-XL to have a pro-apoptotic effect and to induce a conformational change, and the use of the protein TCTP or chemical compounds that mimic the effects of TCTP to induce Bcl-XL to have a pro-apoptotic effect, are described.
2. Description of the Related Art
Apoptosis, also known as programmed cell death, is a physiological process through which the body disposes of unneeded or undesirable native cells. The process of apoptosis is used during development to remove cells from areas where they are no longer required, such as the interior of blood vessels or the space between digits. Apoptosis is also important in the body's response to disease. Cells that are infected with some viruses can be stimulated to undergo apoptosis, thus preventing further replication of the virus in the host organism.
Impaired apoptosis due to blockade of the cell death-signaling pathways is involved in tumor initiation and progression, since apoptosis normally eliminates cells with increased malignant potential such as those with damaged DNA or aberrant cell cycling (White, 1996, Genes Dev, 10:1–15). The majority of solid tumors are protected by at least one of the two cell death antagonists, Bcl-2 or Bcl-XL. Members of the Bcl-2-family are known to modulate apoptosis in different cell types in response to various stimuli. Some members of the family act to inhibit apoptosis, such as Bcl-2 and Bcl-XL, while others, such as BAX, BAK, Bid, and Bad, promote apoptosis. The ratio at which these proteins are expressed can decide whether a cell undergoes apoptosis or not. For instance, if the Bcl-2 level is higher than the BAX level, apoptosis is suppressed. If the opposite is true, apoptosis is promoted. Bcl-2 overexpression contributes to cancer cell progression by preventing normal cell turnover caused by physiological cell death mechanisms, and has been observed in a majority of cancers (Reed, 1997, Sem Hematol, 34:9–19; Buolamwini, 1999, Curr Opin Chem Biol, 3:500–509). The expression levels of Bcl-2 proteins often correlate with resistance to a wide spectrum of chemotherapeutic drugs and γ-radiation therapy. Paradoxically, high levels of Bcl-2 also associate with favorable clinical outcomes for patients with some types of cancers. Therefore, Bcl-2 represents an excellent target for the treatment of cancer, especially those in which Bcl-2 is overexpressed and for which traditional therapy has failed.
Biological approaches targeted at Bcl-2 using antisense oligonucleotides have been shown to enhance tumor cell chemosensitivity. Bcl-2 antisense oligonucleotides in combination with chemotherapy are currently in phase II/III clinical trials for the treatment of patients with lymphoma and malignant melanoma, and further trials with patients with lung, prostate, renal, or breast carcinoma are ongoing or planned (Reed, 1997, supra; Piche et al. 1998 Cancer Res 2134–2140; Webb et al. 1997 Lancet 349:1137–1141; Jansen et al. 1998 Nat Med 4:232–234; Waters et al. 2000 J Clin Oncol 18:1812–1823). Recently, cell-permeable Bcl-2 binding peptides and chemical inhibitors that target Bcl-2 have been developed, and some of them have been shown to induce apoptosis in vitro and in vivo (Finnegan et al. 2001 Br J Cancer 85:115–121; Enyedy et al. 2001 J Med Chem 44:4313–4324; Tzung et al. 2001 Nat Cell Biol 3:183–191; Degterev et al. 2001 Nat Cell Biol 3:173–182).
One well-established apoptotic pathway involves mitochondria (Green and Reed, 1998 Science 281:1309–1312; Green and Kroemer, 1998 Trends Cell Biol 8:267–271). Cytochrome c is exclusively present in mitochondria and is released from mitochondria in response to a variety of apoptotic stimuli. Many Bcl-2-family proteins reside on the mitochondrial outer membrane. Bcl-2 prevents mitochondrial disruption and the release of cytochrome c from mitochondria, while BAX and BAK create pores in mitochondrial membranes and induce cytochrome c release. Recent evidence has indicated, however, that Bcl-2 under certain conditions can function as a pro-apoptotic molecule (Finnegan et al. 2001, supra; Fujita et al. 1998 Biochem Biophys Res Commun 246:484–488; Fadeel et al. 1999 Leukemia 13:719–728; Grandgirard et al. 1998 EMBO J. 17:1268–1278; Cheng et al. 1997 Science 278:1966–1968; Del Bello et al. 2001 Oncogene 20:4591–4595). Bcl-2 can be cleaved by caspase-3 and thus be converted to a pro-apoptotic protein similar to BAX (Cheng et al., 1997, supra). Conversely, BAX has also been shown to inhibit neuronal cell death when infected with Sinbis virus (Lewis et al. 1999 Nat Med 5:832–835). These observations suggest that members of the Bcl-2-family have reversible roles in the regulation of apoptosis and have the potential to function either as a pro-apoptotic or anti-apoptotic molecule.
Members of the Bcl-2-family of proteins are highly related in one or more specific regions, commonly referred to as Bcl-2 homology (BH) domains. BH domains contribute at multiple levels to the function of these proteins in cell death and survival. The BH3 domain, an amphipathic α-helical domain, was first delineated as a stretch of 16 amino acids in Bak that is required for this protein to heterodimerize with anti-apoptotic members of the Bcl-2-family and to promote cell death. All proteins in the Bcl-2-family contain a BH3 domain, and this domain can have a death-promoting activity that is functionally important. The BH3 domain acts as a potent “death domain” and there is a family of pro-apoptotic proteins that contain BH3 domains which dimerize via those BH3 domains with Bcl-2, Bcl-XL and other anti-apoptotic members of the Bcl-2 family. Structural studies revealed the presence of a hydrophobic pocket on the surface of Bcl-XL and Bcl-2 that binds the BH3 peptide. Interestingly, the anti-apoptotic proteins Bcl-XL and Bcl-2 also possess BH3 domains, but in these anti-apoptotic proteins, the BH3 domain is buried in the core of the protein and not exposed for dimerization. (Kelekar and Thompson 1998 Trends Cell Biol 8:324). NMR structural analysis of the Bcl-XL/Bak BH3 peptide complex showed that the Bak BH3 domain binds to the hydrophobic cleft formed in part by the BH1, BH2 and BH3 domains of Bcl-XL (Sattler 1997 Science 275:983; Degterev 2001 Nature Cell Biol 3:173–182). BH3-domain-mediated homodimerizations and heterodimerizations have a key role in regulating apoptotic functions of the Bcl-2-family (Diaz et al. 1997 J Biol Chem 272:11350; Degterev 2001 Nature Cell Biol 3:173–182).
The orphan receptor TR3 (also known as nur77 or nerve growth factor-induced clone B NGFI-B) (Chang and Kokontis 1988 Biochem Biophys Res Commun 155:971; Hazel et al. 1988 PNAS USA 85:8444) functions as a nuclear transcription factor in the regulation of target gene expression (Zhang and Pfahl 1993 Trends Endocrinol Metab 4:156–162; Tsai and O'Malley 1994 Annu Rev Biochem 63:451; Kastner et al. 1995 Cell 83:859; Mageldorf and Evens 1995 Cell 83:841). TR3 was originally isolated as an immediate-early gene rapidly expressed in response to serum or phorbol ester stimulation of quiescent fibroblasts (Hazel et al., supra; Ryseck, et al. 1989 EMBO J. 8:3327; Nakai et al. 1990 Mol Endocrinol 4:1438; Herschman 1991 Annul Rev Biochem 60:281). Other diverse signals, such as membrane depolarization and nerve growth factor, also increase TR3 expression (Yoon and Lau 1993 J Biol Chem 268:9148). TR3 is also involved in the regulation of apoptosis in different cell types (Woronicz et al. 1994 Nature 367:277; Liu et al. 1994 Nature 367:281; Weih et al. PNAS USA 93:5533; Chang et al. 1997 EMBO J. 16:1865; Li et al. 1998 Mol Cell Biol 18:4719; Uemura and Chang, 1998 Endocrinology 129:2329; Young et al. 1994 Oncol Res 6:203). It is rapidly induced during apoptosis of immature thymocytes and T-cell hybridomas (Woronicz et al., supra; Liu et al., supra), in lung cancer cells treated with the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) (Li et al., supra) (also called CD437), and in prostate cancer cells treated with different apoptosis inducers (Uemura and Chang, supra; Young et al., supra). Inhibition of TR3 activity by overexpression of dominant-negative TR3 or its antisense RNA inhibits apoptosis, whereas constitutive expression of TR3 results in massive apoptosis (Weih et al., supra; Cheng et al., supra).
Further studies of TR3 have yielded a better understanding of its mechanism of action in apoptosis (Li et al., 2000, Science 289:1159). First, several apoptosis inducing agents which also induced TR3 expression in human prostate cancer cells were identified. These included the AHPN analog 6-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chloro-2-naphthalenecarboxylic acid (MM11453), the retinoid (Z)-4-[2-bromo-3-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)propenoyl]benzoic acid (MM11384), the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the calcium ionophore A23187, and the etoposide VP-16. Second, it was found that the transactivation activity of TR3 is not required for its role in inducing apoptosis, as demonstrated by an experiment that showed that apoptosis inducing agents blocked the expression of a TR3 target reporter gene. This was further supported by the finding that a TR3 mutant deprived of its DNA binding domain (DBD) was still competent for inducing apoptosis. Third, TR3 was found to relocalize to the outer surface of the mitochondria in response to some apoptotic stimuli. TR3, visualized in vivo by tagging with Green Fluorescent Protein (GFP), was shown to relocalize from the nucleus to the mitochondria in response to apoptosis-inducing agents. Fractionation studies showed that TR3 was associating with the mitochondria-enriched heavy membrane fraction, and proteolysis accessibility studies on purified mitochondria confirmed that TR3 was associating with the outer surface of the mitochondria, where Bcl-2-family members are also found. Fourth, TR3 was shown to be involved in the regulation of cytochrome c release from the mitochondria. Inhibition of TR3 activity by expression of TR3 antisense RNA blocked the release of cytochrome c and mitochondrial membrane depolarization in cells stimulated with TPA and MM11453. Furthermore, incubating purified mitochondria with recombinant TR3 protein resulted in cytochrome c release.
Li et al., 2000, supra, further explored the function of TR3 through mutation of the protein. A TR3 mutant which had the DNA binding domain (amino acid residues 168–467) removed (TR3/ΔDBD) no longer localized in the nucleus in non-stimulated cells, but instead was consistently found in mitochondria. This localization phenotype was accompanied by a constant release of cytochrome c from the mitochondria. Three other deletion mutants were also generated and assayed: an amino-terminal deletion of 152 amino acids referred to as TR3/Δ1, a 26 amino acid carboxy-terminal deletion referred to as TR3/Δ2, and a 120 amino acid carboxy-terminal deletion referred to as TR3/Δ3. The TR3/Δ1 protein did not relocalize to the mitochondria in response to TPA, but maintained a nuclear localization. TR3/Δ1 had a dominant negative effect, preventing the relocalization of full-length TR3 to the mitochondria and inhibiting apoptosis. Mitochondrial targeting was still observed in TR3/Δ2 protein expressing cells, but not in TR3/Δ3 protein cells in response to TPA treatment. These results indicated that carboxy-terminal and amino-terminal sequences are crucial for mitochondrial targeting of TR3 and its regulation.
Experiments designed to alter the localization of TR3/ΔDBD by fusing it to various cellular localization signals showed that TR3 must have access to the mitochondria in order to induce its pro-apoptotic effect. When TR3/ΔDBD was fused to a nuclear localization sequence, a plasma membrane targeting sequence, or an ER-targeting sequence, TR3/ΔDBD was not targeted to the mitochondria and no induction of cytochrome c release was observed.
The translationally controlled tumor-associated protein (TCTP) is conserved across a wide range of eukaryotes and shows no significant sequence homology with any other proteins. The precise function of the family remains elusive. TCTP has been described as growth related protein. TCTP was originally identified as a serum-inducible 23-kDa protein band that undergoes an early and prominent increase upon serum stimulation in tissue culture cells (Benndorf et al. 1988 Exp Cell Res 174:130). TCTP mRNA is expressed at constant levels in both growing and nongrowing cells, and the translation is regulated by its polypyrimidine-rich 5′ untranslated region (Bohm et al. 1991 Biomed Biochim Acta 50:1193; 174:130). TCTP was shown to be one of the first proteins to be induced in Ehrlich ascites tumor cells following mitotic stimulation (Bohm et al. 1989 Biochem Int 19:277) and has been found to be amongst a small group of Schizosaccharomyces pombe proteins that are repressed in response to conditions that arrest cell growth, such as ammonium starvation (Bonnet C. et al. 2000 Yeast 16:23). TCTP was recently shown to be a tubulin-binding protein that dynamically interacts with microtubules during the cell cycle. In addition, TCTP levels in overexpressing cells were correlated with microtubule stabilization and reduced growth rate in vivo (Gachet et al. 1999 J Cell Sci 112:1257). The expression of TCTP also appears to be regulated at two distinct levels in response to the concentration of calcium in different cellular compartments. Whereas depletion of the ER store causes an increase in TCTP mRNA abundance, increased cytosolic calcium concentrations regulate gene expression at the post-transcriptional level (Xu et al. 1999 Biochem J342:683). The solution structure of TCTP forms a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP-free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (Thaw et al. 2001 Nat Struct Biol 8:701).
The identification of compounds having the ability to alter the activity of Bcl-2-family members from anti-apoptotic to pro-apoptotic would have important therapeutic applications, for example, in the treatment of cancer and other diseases.